The long term objective of this project is to examine the mechanisms of tumor induction by aromatic amines. Derivatives of aromatic amines are known human carcinogens and are extensively employed in chemical, food and pharmaceutical industries presenting potential danger of exposure. The specific aim of this proposal is to examine single and site specific mutagenesis induced by aromatic amines (N-hydroxy-2-aminofluorene, N-acetoxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene and N-acetoxy-N-trifluoroacetyl-2-aminofluorene) at the nucleotide sequence level. The experimental protocol involves the use of Messing's M13 lac hybrid phage and its bacterial host E. coli. First, multiple DNA adducts will be introduced in a part of the lac gene of the phage through in vitro DNA manipulation. Mutants will be isolated after transfection into competent E. coli cells and a representative mutants will be analyzed at the sequence level to answer the following questions: 1) What are the principal types of mutations (point, frameshift, insertion or deletion) produced? If there are deletion or insertion, how many bases are involved? 2) What are the sites of mutations, random or nonrandom? If they are nonrandom, are these sites located within a specific region or a specific sequence pattern? 3) Is there any correlation between aminofluorene-guanine adduct site and the mutation? On the basis of the above results, single and site specific modification will be introduced in the DNA and its mutational consequences will be examined as above. Essentially we will address to questions similar as before for multiple adducts. Mutational studies will be complemented by investigations into the area of DNA damage by these aromatic amines derivatives. Experiments will be performed to determine any patterns of DNA damage in vitro and in vivo and its relation to the mutagenic consequences.